Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips

Abstract SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.


Fabrication of microfluidic devices
The microfluidic device consists of two layers of microchannels separated by two semipermeable membranes (Fig. 1A). The microchannel layers were fabricated from PDMS using a soft lithographic method (1). PDMS prepolymer (Sylgard 184, Dow Corning) at a ratio of 10:1 base to curing agent was cast against a mold composed of SU-8 2150 (MicroChem) patterns formed on a silicon wafer. The cross-sectional size of the microchannels was 1 mm in width and 300 μm in height. To introduce solutions into the microchannels, access holes were punched through the PDMS using a 6-mm biopsy punch (Kai Corporation). Two PDMS layers were bonded to semipermeable PET membranes containing 3.0 μm pores (#353091, Corning) using a thin layer of liquid PDMS prepolymer as the mortar (2). PDMS prepolymer was spin-coated (4,000 rpm for 60 sec) onto a glass slide. Subsequently, both the top and bottom channel layers were placed on the glass slide to transfer the thin layer of PDMS prepolymer onto the embossed PDMS surfaces. The two PET membranes were placed on the top layer and then bonded to the bottom layer. The combined layers were left at room temperature for 1 day to remove air bubbles and then put into an oven at 60°C overnight to cure the PDMS glue.
The PDMS-based microfluidic devices (PDMS devices) were sterilized by placing them under UV light for 1 hr prior to the cell culture.

SARS-CoV-2 infection and drug treatment
SARS-CoV-2 (0.1 MOI) containing Hepatocyte Culture Medium BulletKit (HCM, Lonza) was injected into the top channel of ibd-and bv-LoCs. After 1 dpi (days post-infection), the culture medium was replaced with fresh medium and, the ibd-and bv- CCGCCATTGCCAGCCATTC. Standard curves were prepared using SARS-CoV-2 RNA (10 5 copies/μL) purchased from Nihon Gene Research Laboratories.

Lactate dehydrogenase (LDH) release
To evaluate LDH release, the culture supernatants of the top and bottom channels of the ibd-and bv-LoCs were collected. The collected supernatants were analyzed using the LDH-Glo Cytotoxicity Assay (Promega) according to the manufacturer's instructions.

Evaluation of transport activity of bile acid, bilirubin, and albumin
To evaluate the transport activity of bile acids, the culture supernatants of the top and bottom channels of the ibd-and bv-LoCs, which were cultured for 24 hr, were collected. The collected supernatants were analyzed using the Total Bile Acid Assay Kit (Cell Biolabs) according to the manufacturer's instructions.
To evaluate the transport activity of direct bilirubin, 10 μM bilirubin was injected into the top channel. After 48 hr, the culture supernatants of the top and bottom channels were collected. The collected supernatants were analyzed using the QuantiChrom Bilirubin Assay Kit (BioAssay Systems) according to the manufacturer's instructions.
To evaluate the transport activity of albumin, the culture supernatants of the top and bottom channels were collected at 4 days after the hepatocyte seeding. The collected supernatants were analyzed using the Human Albumin ELISA Quantitation Set (Bethyl Laboratories). ELISA was performed according to the manufacturer's instructions. hr.

BODIPY staining
The ibd-and bv-LoCs were fixed with 4% paraformaldehyde in PBS for 15 min and then incubated with 10 μg/mL BODIPY 493/503 (Thermo Fisher Scientific) for 30 min.

Figure S3 SARS-CoV-2 infection efficiency of hepatocytes in ibd-and bv-LoCs
The ibd-and bv-LoCs were infected with 0.1 MOI SARS-CoV-2. At 2, 7, and 14 dpi, the viral RNA copy number in the cell culture supernatant in the top channel (A) or bottom channel (B) was measured by qPCR. Data are shown as means ± SD (n=3, technical replicates).
A volcano plot of differentially expressed genes between mock and infected hepatocytes (log2 fold-change > 2, adjusted p-value (padj) < 0.01). Orange dots represent up-regulated genes and blue dots represent down-regulated genes.

SARS-CoV-2 infection in hepatocytes of bv-LoCs
The